Replication of HAV is being studied in order to find ways to improve the growth characteristics of the virus, develop new attenuated vaccine strains, and analyze the potential use of HAV as a vector for gene therapy. As the virus presently grows slowly and to relatively low titer, vaccine production is inefficient. Several different constructs using the infectious cDNA of HAV as the backbone have been made. We have inserted a multiple cloning site and the cleavage site for 3Cpro just prior to the viral polyprotein initiation site into the infectious cDNA of HAV. As a demonstration. we have now inserted synthetic oligonucleotides coding for an octapeptide, "FLAG peptide" and shown that this construct was both infectious. Using a monoclonal antibody against Flag, specific expression was demonstrated. Similarly, a nonpeptide coding for the "a" epitope of the hepatitis B surface antigen (HBsAg) was molecularly cloned into the infectious cDNA of HAV and infectious virus was derived upon transfection of transcripts into FRhK/4 cells. Further modifications in the constructs are being made in order to express larger polypeptides with the aim of being able to express immunenhancers such as cytokines as part with the virus thus improving immunogenicity. As HAV is a positive stranded RNA virus with no potential for integration and as it does not produce a cytopathic effect of chronic infections, the virus has some ideal characteristics for gene therapy.